Glioblastoma multiforme (GBM), the most common primary brain tumor in adults, is associated with a dismal prognosis of only 12-15 months despite aggressive surgery, radiation, and chemotherapy. The lack of effective treatment options has made this disease a target for new strategies such as gene therapy. However, the only major Phase III clinical trial of gene therapy, involving the use of conventional replication-defective retrovirus vectors in GBM patients, resulted in disappointingly low and therapeutically inadequate transduction levels on the order of only 0.02%. The inability of standard replication-defective retroviral vectors to achieve effective transduction of tumors in vivo is therefore a major obstacle to gene therapy for gliomas. The use of replication-competent vectors for gene transfer would be more efficient, as each tumor cell that is successfully transduced would itself become a virus-producing cell, sustaining further transduction events even after initial administration. We have previously demonstrated that direct intratumoral injection of murine leukemia virus (MLV)-based replication-competent retrovirus (RCR) vector preparations can achieve tremendously efficient suicide gene transfer in gliomas, with transduction stringently restricted to the actively dividing tumor cells without evidence of significant spread to extratumoral sites, and resulting in significantly prolonged survival upon prodrug administration, without detectable systemic side effects. In collaboration with neurosurgery groups at UCLA, USC, and UCSF, and the National Gene Vector Laboratory (NGVL), here we propose to develop and implement clinical grade RCR vector production (Aim 1), to validate these clinical grade vectors by confirmatory testing of therapeutic efficacy in rodent intracranial glioma models, and in larger canine models (Aim'2), to optimize monitoring methodologies as mandated by FDA guidelines, and to develop clinical trial protocols (Aim 3). Hence, we propose to perform these necessary preclinical translational studies through this U01 mechanism, with the final goal of filing an IND and obtaining approval from the FDA to initiate clinical trials.